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An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development

ABSTRACT: Rag1 and Rag2 gene expression in CD4+CD8+ double positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes. Sequencing of Satb1-ChIP and input control from 6 wk old thymus

ORGANISM(S): Mus musculus

SUBMITTER: Hunter Richards

PROVIDER: E-GEOD-66248 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 and Rag2 gene expression during thymocyte development

Project description:Rag1 and Rag2 gene expression in CD4+CD8+ double positive (DP) thymocytes depends on the activity of a distant anti-silencer element (ASE) that counteracts the activity of an intergenic silencer. However, the mechanistic basis for ASE activity is unknown. Here we show that the ASE physically interacts with the distant Rag1 and Rag2 gene promoters in DP thymocytes, bringing the two promoters together to form an active chromatin hub. Moreover, we show that the ASE functions as a classical enhancer that can potently activate these promoters in the absence of the silencer or other locus elements. In thymocytes lacking the chromatin organizer SATB1, we identified a partial defect in Tcra gene rearrangement that was associated with reduced expression of Rag1 and Rag2 at the DP stage. SATB1 binds to the ASE and Rag promoters, facilitating inclusion of Rag2 in the chromatin hub and the loading of RNA polymerase II to both the Rag1 and Rag2 promoters. Our results provide a novel framework for understanding ASE function and demonstrate a novel role for SATB1 as a regulator of Rag locus organization and gene expression in DP thymocytes.

2015-02-25 | GSE66248 | GEO

Chromatin organizer SATB1 controls the cell identity of CD4+ CD8+ double-positive thymocytes by compacting super-enhancers

Project description:CD4+ and CD8+ double-positive (DP) thymocytes are at a critical stage during the T cell development in thymus. DP cells rearrange the T cell receptor gene Tcra to generate T cell receptors with TCR. Then DP cells differentiate into CD4 or CD8 single-positive (SP) thymocytes, Regulatory T cells, or invariant nature kill T cells (iNKT) according to the TCR signal. Chromatin organizer SATB1 is highly expressed in DP cells and plays an essential role in regulating Tcra rearrangement and differentiation of DP cells. Here we explored the mechanism of SATB1 orchestrating gene expression in DP cells. Single-cell RNA sequencing assay of SATB1-deficient thymocytes showed that the cell identity of DP thymocytes was changed, and the genes specifically highly expressed in DP cells were down-regulated. ChIP-seq and ATAC-seq data showed the similar tendency. The super-enhancers regulate the expressions of the DP-specific genes, and the SATB1 deficiency reduced the super-enhancer activity. Hi-C data showed that interactions in super-enhancers and between super-enhancers and promoters decreased in SATB1 deficient thymocytes. We further explored the regulation mechanism of two SATB1-regulating genes, ETS2 and Bcl6, in DP cells and found that the knockout of the super-enhancers of these two genes impaired the development of DP cells. Our research reveals that SATB1 globally regulates super-enhancers of DP cells and promotes the establishment of DP cell identity, which helps understand the role of SATB1 in thymocyte development.

2021-09-02 | GSE182995 | GEO

RAG Represents a Widespread Threat to the Lymphocyte Genome - (ChIP-seq)

Project description:The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mistargeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report the surprising finding that RAG1 binds to thousands of sites in the genome of developing lymphocytes, primarily at active promoters and enhancers. The genome has responded by reducing the abundance of "cryptic" recombination signals near sites of RAG1 binding. This depletion operates specifically on the RSS heptamer, with nonamers enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes. RAG1,RAG2 and H3K4me3 ChIP-seq profiles of human thymocytes, mouse thymocytes and preB cells, and Abelson pre-B cell line treated with STI-571

2015-08-06 | E-GEOD-69475 | biostudies-arrayexpress

RAG1 targeting in the genome is dominated by chromatin interactions mediated by the non-core regions of RAG1 and RAG2

Project description:The RAG1/RAG2 endonuclease initiates V(D)J recombination at antigen receptor loci but also binds to thousands of places outside of these loci. RAG2 localizes directly to lysine 4 trimethylated histone 3 (H3K4me3) through a PHD finger. The relative contribution of RAG2-dependent and RAG1-intrinsic mechanisms in determining RAG1 binding patterns is not known. Through analysis of deep RAG1 ChIP-seq data, we provide a quantitative description of the forces underlying genome-wide targeting of RAG1. Surprisingly, sequence-specific DNA binding contributes minimally to RAG1 targeting outside of antigen receptor loci. Instead, RAG1 binding is driven by two distinct modes of interaction with chromatin: the first is driven by H3K4me3, promoter-focused, and dependent on the RAG2 PHD, and the second is defined by H3K27Ac, enhancer-focused, and dependent on "non-core" portions of RAG1. Based on this and additional chromatin and genomic features, we formulated a predictive model of RAG1 targeting to the genome. RAG1 binding sites predicted by our model correlate well with observed patterns of RAG1-mediated breaks in human pro-B acute lymphoblastic leukemia. Overall, this study provides an integrative model for RAG1 genome-wide binding and off-target activity, and reveals a novel role for the RAG1 non-core region in RAG1 targeting. ChIP-seq profiles of RAG1 from mouse thymocytes, and H3K27Ac from human REH cell line

2016-07-06 | E-GEOD-84052 | biostudies-arrayexpress

The In Vivo Pattern of Binding of RAG1 and RAG2 to Antigen Receptor Loci

Project description:The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing IgÎº and TcrÎ± J gene segments and Igh and TcrÎ² J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding is restricted to regions containing recombination signal sequences, RAG2 binds extremely broadly in a pattern that mirrors that of trimethylated lysine 4 of histone 3. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination. RAG2 binding was analyzed in wild type, RAG2-/-Î², and D708A-RAG1-/-Î² thymocytes. Histone modification H3K4me3 was analyzed in D708A-RAG1-/-Î² and wild type thymocytes.

2010-04-19 | E-GEOD-21207 | biostudies-arrayexpress

RAG Represents a Widespread Threat to the Lymphocyte Genome - (Mnase-seq)

2015-08-06 | E-GEOD-69474 | biostudies-arrayexpress

Gene expression analysis of CD4+ CD8+ thymocytes [Affymetrix]

Project description:We performed ChIP-Seq for hallmark TFs (Ets1, Runx1), histone modification marks (H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3), total RNA Pol II, short RNA-Seq as well as nucleosome mapping mainly in murine Rag2 -/- thymocytes. We also performed ChIP-Seq for E47 as well as nucleosome mapping, gene expression microarray analysis in CD4+ CD8+ DP thymocytes. Overall, we find a key role for the transcription factor Ets1, contributing towards alpha beta T cell lineage commitment via differential transactivation of stage-specific genes orchestrated by dynamic, co-association -mediated chromatin remodeling, as well as transcription dependent generation of a specialized chromatin structure at the TCR beta locus. Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes Genome-wide analysis via ChIP-Seq for Ets1, Runx1, total RNA Pol II binding, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, short RNA-Seq, Mnase-Seq in murine Rag2 -/- thymocytes, ChIP-Seq for E47, Mnase-Seq and gene expression microarray analysis in DP thymocytes This Series represents gene expression microarray data.

2014-04-01 | E-GEOD-56357 | biostudies-arrayexpress

Satb1 Overexpression Drives Tumor-Promoting Activities In Cancer-Associated Dendritic Cells

Project description:Special AT-rich sequence-binding protein-1 (Satb1) governs genome-wide transcriptional programs. Using a new conditional knockout mouse, we found that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating MHC-II expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy of the H2-Ab1 promoter, which activates MHC-II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46+ inflammatory DCs that universally infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. Correspondingly, specific in vivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but relentless Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression. RNA-seq with whild type and knocked-down Satb1

2016-01-13 | E-GEOD-76776 | biostudies-arrayexpress

Deeply hidden genome organization mediated by base-unpairing regions (BURs) directly bound by SATB1 linked to transcription

Project description:Mammalian genomes are organized by multi-layered chromatin folding. How three-dimensional genome organization contributes to cell-type specific transcription remains unclear. We uncover genomic elements termed base-unpairing regions (BURs), distributed genome-wide, as the sole and direct targets of cell-type specific SATB1 protein in vivo. The SATB1 direct-binding profile was generated by analyzing stringently-purified genomic DNA crosslinked to its directly-bound proteins only (ureaChIP-seq). Furthermore, a SATB1-bound BUR interacts extensively and frequently over the entire 5.7 megabase gene-rich region within many regulatory regions, including those near SATB1-dependent Rag1/Rag2 genes. SATB1 depletion leads to major loss of these interactions with greatly reduced Rag1/Rag2 expression. Most BURs reside within lamina associated domains (LADs), among which SATB1 binds to cell-type specific groups of BURs. Genome organization mediated by CTCF and SATB1 are distinct as these proteins do not co-bind chromatin in vivo and their direct binding sites are mutually exclusive genome-wide. These results revealed a previously undetected chromatin organization mediated by SATB1 direct binding to selected BURs genome-wide and suggest that chromatin interactions from some of these BURs provide a regulatory network underlying cell-type specific gene expression.

2021-12-28 | GSE191146 | GEO

RNAseq analysis of thymic CD4-CD8- double-negative (DN) invariant NKT cells from CD4+CD8+ double-positive (DP)-specific Rag2-conditional knock-out mice

Project description:We report the identification of DN NKT cells developed from DN stage thymocytes. We analyzed the gene expression profiles of NKT cells that have developed from DN stage thymocytes isolated from DP-specific E8III-Cre transgenic Rag2-floxed mouse strain, and NKT cells developed from DP thymocytes sorted as YFP-reporter positive NKT cells that were isolated from E8III-Cre transgenic Rosa26-loxP-STOP-loxP mouse.

2016-11-30 | GSE89532 | GEO

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